1. Field of the Invention
This invention relates to hybridoma cell lines and monoclonal antibodies produced therefrom which may be used to detect tilmicosin.
2. Description of the Prior Art
Tilmicosin is a semi-synthetic macrolide antibiotic that is approved for veterinary use in cattle and swine to combat respiratory disease in the US, and in Canada for the treatment of pneumonia in lambs. Bovine respiratory disease associated with Mannheimia (Pasteurella) haemolytica can be treated and controlled by a single subcutaneous injection of tilmicosin. Tilmicosin also has in vitro activity against several gram-negative bacteria associated with respiratory disease, including Actinobacillus pleuropneumoniae, Pasteurella multocida, P. haemolytica, and Mycoplasma spp. The administration of tilmicosin may also be a convenient way to reduce mastitis infection.
Tilmicosin is derived from the macrolide antibiotic tylosin, produced by Streptomyces fradiae. Synthesis of tilmicosin was described by Debono et al. (U.S. Pat. No. 4,820,695). Briefly, in a two step process, tylosin is first hydrolyzed to yield desmycosin, which is then treated with dimethyl piperidine to produce tilmicosin.
It is known that most antimicrobial agents have limited ability for cellular penetration. However, tilmicosin has been shown to concentrate in bovine lung tissue. Even after serum levels have dropped below therapeutic concentrations, lung concentrations have been shown to exceed the P. haemolytica MIC for at least 72 hours after dosage.
Attempts have been made to produce a quick and economical competitive enzyme-linked immunosorbent assay (cELISA) method for determining tilmicosin residues in feeds and tissues. Jackman et al. (1997, J. Vet. Pharmacol. Therap., 20 (suppl. 1):131-132) reported the preparation of polyclonal antibodies to tilmicosin using desmycosin and lactenocin conjugated to carrier proteins. Silverlight et al. (1999, Food and Agricultural Immunology, 11:321-328) later reported that the antibodies raised against desmycosin and lactenocin conjugates bound both tilmicosin and tylosin. However, tilmicosin could not be used in an immunogenic preparation.
Despite these advances, the need persists for a monoclonal antibody to tilmicosin having improved sensitivity and specificity.
We have now discovered hybridoma cell lines which produce and secrete monoclonal antibodies which selectively bind to tilmicosin. We have unexpectedly found that these hybridomas may be obtained by using as an immunization agent or immunogen, 23-deoxo-23-demycinosyl tilmicosin which has been conjugated to an immunogenic carrier. Tilmicosin in biological samples may be detected and quantified by contacting the sample with the antibodies to form a tilmicosin/antibody immunocomplex when tilmicosin is present, which immunocomplex may then be detected. The monoclonal antibodies also may be incorporated into kits for the detection and quantification of tilmicosin.
It is an object of this invention to provide hybridoma cell lines that produce and secrete high affinity monoclonal antibodies which selectively bind to tilmicosin.
A further object of this invention is to provide monoclonal antibodies which selectively bind to tilmicosin but not tylosin.
Another object of this invention is to provide immunoassay methods for the measurement of tilmicosin in biological samples.
A further object is to provide kits useful for the assay of tilmicosin which include the monoclonal antibodies described herein.
Yet another object is to provide an immunization agent which may be used to produce hybridoma cell lines that produce and secrete high affinity monoclonal antibodies which selectively bind to tilmicosin.
Other objects and advantages of this invention will become readily apparent from the ensuing description.
In accordance with this invention we have created hybridoma cell lines that produce monoclonal antibodies that bind tilmicosin and are effective for detecting and quantifying levels of this antibiotic. We have unexpectedly discovered that by use of a novel immunogen, monoclonal antibodies may be produced which possess improved specificity and increased affinity for tilmicosin. The antibodies of this invention may be used to rapidly and accurately detect and quantify tilmicosin, providing an indicator of the level of this antibiotic in biological samples.
Traditionally, preparation of hybridomas may be accomplished using conventional techniques such as described by Kohler and Milstein [Nature, 256:495-497 (1975)], Koprowski et al. [U.S. Pat. No. 4,196,265], Wands [U.S. Pat. No. 4,271,145], or Stanker et al. [U.S. Pat. No. 5,466,784], the contents of each of which are incorporated by reference herein. Briefly, the process of preparation comprises the steps of immunizing an animal with the antigen of interest, recovering splenocytes or lymphocytes from the animal, fusing the splenocytes or lymphocytes with continuously replicating myeloma cells to produce hybrid cells, and screening the resultant hybrid cells for the production of antibodies to the antigen.
Often, the compound of interest is a relatively small molecule, and hence is itself incapable or only poorly capable of stimulating the immune system to produce antibodies. To render such compounds immunogenic, they are generally conjugated to an immunogenic carrier in such a manner that the resultant immunogen is capable of stimulating the immune system of an animal to produce specific antibodies that are capable of binding the unconjugated compound. Application of this traditional protocol for the generation of monoclonal antibodies to a small compound such as tilmicosin, would logically dictate an immunogen prepared by conjugation of tilmicosin to a carrier protein. However, in a departure from established practice, we describe here the preparation of monoclonal antibodies using significantly different, novel immunogens.
The method of preparing the hybridomas comprises the following steps:
Immunogen. The immunization agent of this invention is not constructed from tilmicosin, but is derived from 23-demycinosyl tilmicosin (which may also be referred to as 20-deoxo-20-[3,5-dimethyl-piperidin-1-yl]desmycosin). The structures of tilmicosin and 23-demycinosyl tilmicosin are shown in formulas I and II, respectively: 
The immunization agent is prepared by covalently conjugating an immunogenic carrier to 23-deoxo-23-demycinosyl tilmicosin at carbon atom number 23 thereof. Immunogenic carriers are defined herein as any compound to which the haptens may be attached to render them immunogenic. Suitable carriers are well known and may be readily determined by the practitioner skilled in the art. Without being limited thereto, preferred carriers include proteins such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, glucose oxidase, and human thyroglobulin.
The immunogenic carrier may be conjugated to the 23-deoxo-23-demycinosyl tilmicosin molecule directly or through an optional crosslinking agent or spacer. In accordance with the preferred embodiment, the immunogen is created by conjugating the carrier to 23-deoxo-23-demycinosyl tilmicosin which has been modified by reaction at the C23 thereof with a crosslinking agent. A variety of diamine crosslinking agents are suitable for use herein, with diamino hydrocarbons and/or thiols being preferred. Suitable diamino hydrocarbons include, but are not limited to straight or branched chain, cyclic, saturated or unsaturated, diamino hydrocarbons, such as 1,3-diaminopropane, 1,4-diaminobutane, or hexamethylenediamine.
In a particularly preferred embodiment which is described in greater detail in Example 1, to selectively bind the crosslinking agent to C23 of the hapten, the C23 of 23-demycinosyl tilmicosin (II) is first halogenated, for example, by treatment with Cl2, F2, or Br2, and preferably I2. In this initial reaction, only the C23 is halogenated to a significant degree, with the hydroxyl group of 23-demycinosyl tilmicosin being displaced by the halogen to yield 23-halo-23-deoxo-23-demycinosyl tilmicosin of the formula (III): 
The other hydroxyl groups of the 23-demycinosyl tilmicosin are not displaced to any significant degree. Following this displacement reaction, (III) may then be reacted with the diamino hydrocarbon H2Nxe2x80x94Rxe2x80x94NH2, with the halide being in turn displaced by the amine, yielding 23-diamino-23-deoxo-23-demycinosyl tilmicosin of the formula (IV): 
The carrier protein may be directly conjugated to the terminal amine of (IV) using techniques known in the art. However, in the preferred embodiment, the terminal amine of (IV) is first displaced with a thiol such as described in Example 1, which may be more readily conjugated to the carrier protein.
In an alternative reaction, it is also envisioned that the carrier may be conjugated directly to 23-deoxo-23-demycinosyl tilmicosin without use of a crosslinking agent. For instance, without being limited thereto, 23-halo-23-deoxo-23-demycinosyl tilmicosin (III) may be reacted with carrier protein, with the halide being displaced by an amine of the protein.
In yet another alternative embodiment, 23-thiol-23-deoxo-23-demycinosyl tilmicosin may be prepared by displacement of the halide of (III) by a thiol, which thiol may then be conjugated to the carrier protein. In this embodiment, the thiol is effective as an alternative crosslinking agent.
Immunogens of this invention prepared by conjugation of 23-deoxo-23-demycinosyl tilmicosin to a carrier protein through a crosslinking agent may be generally described by the formula (V): 
wherein L refers to the optional crosslinking agent, and Rxe2x80x2 is a carrier protein.
Immunization. To generate antibody-producing splenocytes or lymphocytes, an immunizing preparation comprising the 23-deoxo-23-demycinosyl tilmicosin-carrier complex is injected into an immunologically competent animal. The preparation may also contain other proteins, although pure or partially pure compositions of the conjugate in a pharmaceutically acceptable carrier are preferred.
Without being limited thereto, rats and particularly mice are preferred animals for immunization because of ease of handling. Preparation of hybridomas using splenocytes from these animals fused to a variety of myeloma cell lines have been reported by many investigators.
Inoculations of the animal can be by various routes. A series of three inoculations, generally at two week intervals, with a composition of the complex in isotonic saline with RIBI adjuvant (Immunochem Research, Inc., Hamilton, Mont.) elicits good antibody response, and is preferred. The skilled practitioner will recognize that other routes of administration, immunization schedules, and carriers or adjuvants may be used.
Hybridization. Splenocytes or lymphocytes recovered from the immunized animal are fused with continuously replicating tumor cells, such as myeloma or lymphoma cells, cultured, and hybridoma cells selected. Many continuously replicating tumor cell lines are available which may be used as fusion partners with the splenocytes. Without being limited thereto, preferred myeloma cells include P3-NS1-K653, and particularly SP2/O.
Fusion and culture of the cells can be performed using conventional techniques. In accordance with one well-known effective procedure, the splenocytes and myeloma cells are fused by exposure to polyethylene glycol. Hybrid cells are selected by culture in hypoxanthine-aminopterin-thymidine (HAT) medium, whereby unfused myeloma cells are killed by HAT and splenocytes die out, leaving only the hybrid cells. The resultant hybridomas are then grown in HAT or other suitable culture medium and assayed for antibody production.
Screening. Samples of the supernatant culture fluid from the hybridomas are screened for antibodies to tilmicosin. In accordance with the preferred embodiment, the supernatants are screened using a modification of the direct-binding ELISA (db-ELISA). In this embodiment, solid substrates, such as beads or the wells of a microtiter plate which have been coated with tilmicosin-, or most preferably, 23-deoxo-23-demycinosyl tilmicosin-carrier complex, are used to bind anti-tilmicosin antibody in the supernatants, and bound antibody is then detected.
Following contact of the supernatant culture fluid with the tilmicosin or 23-deoxo-23-demycinosyl tilmicosin coated substrate, detection of bound antibody may be accomplished by addition of enzyme-labeled anti-immunoglobulin antibodies followed by enzyme substrate. While a variety of enzyme/substrate labels may be used, horse radish peroxidase and its substrate, 2,2xe2x80x2-azinobis-3-ethylbenthiazolinesulfonic acid (ABTS) are preferred. In the alternative, it is understood that the supernatants also may be screened using non-enzyme labels, such as radiolabels or chromophores, in related solid-phase immunosorbent techniques such as RIA and FIA.
Cloning. Cloning of hybridomas which are positive for desired antibody production can be carried out as soon as they are detected by any method known in the art. Hybridomas having a positive response in the ELISA screen are preferably expanded and subcloned one or more times by limiting dilution to assure monoclonality.
The supernatant culture fluid from the cloned hybridomas also may be screened to select for those producing antibodies having a high affinity for tilmicosin. Affinity may be measured using solid phase immunoassays such as ELISA, RIA, or equilibrium dialysis using labeled tilmicosin. In the preferred embodiment, affinity is measured by competitive indirect ELISA as described in the Examples, and is conducted at a final antibody concentration (dilution from the tissue culture supernatant) to give 50% of maximal binding to a tilmicosin- or 23-deoxo-23-demycinosyl tilmicosin-carrier complex coated substrate or assay well (i.e., the concentration of the antibody that results in 50% of the plateau activity in direct binding ELISA). In accordance with this embodiment, the antibody containing supernatant is added to a tilmicosin or 23-deoxo-23-demycinosyl tilmicosin coated substrate such as the wells of a microtiter plate (prepared as described above), together with a range of concentrations of free tilmicosin as a competitor. Following incubation and washing, bound antibody in the wells is determined in the same manner as the db-ELISA. Percent inhibition may be calculated as (1xe2x88x92B/Bo)xc3x97100, where B is the optical density (OD) of a well with a competitor and Bo is the mean OD of the wells without competitor (control). The relative affinity of the antibodies may be accurately measured as the concentration of tilmicosin added to the wells that results in at least 20% inhibition (IC20) of control activity. However, for greater accuracy, the affinity may be alternatively measured at 50% inhibition (IC50). In addition to screening the hybridomas for those producing monoclonal antibodies having a high affinity for tilmicosin, the hybridomas may also be screened to select those producing antibodies which do not significantly bind to the related antibiotic, tylosin. As defined herein, antibodies which do not significantly bind to tylosin are those which do not exhibit any observable binding to tylosin at levels of 1,000 ng/well. Alternatively, although the hybridomas described in the Examples produce antibodies which do not bind to tylosin, it is envisioned that hybridomas produced in accordance with this invention may be screened for those producing antibodies which do bind to tylosin.
Once hybridomas producing and secreting the desired anti-tilmicosin antibodies are identified, large quantities of the antibodies may be produced in tissue culture using well-known techniques. In a preferred embodiment, the antibodies are produced in vitro in modular mini fermentors, as described by Falkenberg et al. (1995, J. Immunol. Methods, 179:13-29; and 1998, Res. Immunol., 149:560-570). Alternatively, antibodies may be produced within host animals, such as by ascites formation in syngenic mice. Monoclonal antibodies so produced may be purified, for example, by affinity chromatography on a protein A or G resin, or using tilmicosin bound to a resin.
The monoclonal antibodies produced in accordance with this invention possess high affinity for tilmicosin, allowing the rapid determination of these agents at low levels. As described in detail in the Examples, when the sensitivity was measured at a high standard of accuracy (IC20) by competitive inhibition ELISA, the detection limits of the antibodies for tilmicosin was as low as 0.85 ng.
The antibodies may be used to detect and/or quantify tilmicosin in unknown samples using a variety of conventional immunosorbent assays including but not limited to RIA, FIA or ELISA. A competitive inhibition ELISA similar to that used to screen the hybridomas is preferred. In this assay, a sample to be analyzed is incubated with the monoclonal antibody for tilmicosin and a solid substrate coated with tilmicosin, tilmicosin-carrier complex, 23-deoxo-23-demycinosyl tilmicosin, 23-deoxo-23-demycinosyl tilmicosin-carrier complex, 3,5-dimethyl piperidine, or 3,5-dimethyl piperidine-carrier complex. The hapten coated substrate and any free tilmicosin in the sample thus compete for binding with the antibody. After incubation, the solid phase is drained and washed, and bound antibody on the substrate is detected and percent inhibition calculated as described earlier. The concentration of tilmicosin in the sample may then be determined by reference to a standard curve constructed from assays using known levels of tilmicosin.
In one alternative embodiment, tilmicosin may be determined by a competition ELISA such as described in Brandon et al. (U.S. Pat. No. 5,053,327, the contents of which are incorporated by reference herein) using the monoclonal antibody of the invention attached to a solid support. For example, the anti-tilmicosin antibody may be immobilized on a solid support such as a bead or microtiter well. The unknown sample to be analyzed (or analytical standards of tilmicosin) are then added with enzyme or radiolabeled tilmicosin, and the amount of labeled tilmicosin bound to the antibody is measured, using a substrate when the label is an enzyme. The amount of tilmicosin in the sample is inversely proportional to the amount of bound labeled tilmicosin. In another alternative, the monoclonal antibody may be attached to a solid support for use in conventional double-antibody sandwich ELISA procedures.
With any of the above-described assay formats, the monoclonal antibodies of the invention may be incorporated into kits, alone or preferably together with any other necessary reagents. A preferred kit for use herein comprises a first container including the monoclonal antibody, a second container including detection means effective for detecting bound antibody, and a solid phase support having tilmicosin or 23-deoxo-23-demycinosyl tilmicosin attached thereto.
Determination of tilmicosin in a variety of feeds or biological samples, including animal tissue and animal fluids such as serum, may be conducted using the above-described assays with minimal sample preparation and using simple extraction procedures. For the analysis of tissue samples, the tissue may be homogenized in buffer, such as Tris-HCl, centrifuged, and the liquid phase recovered and used directly in the immunoassay. alternatively, tissue may be probed directly by application of labeled antibody onto the tissue to bind any tilmicosin therein or thereon, and antibody-tilmicosin complex detected as described above. Although any animal tissue may be analyzed, the assay is particularly valuable for the determination of tilmicosin in meats. Tissue for analysis in accordance with the invention may originate from virtually any animal. Without being limited thereto, the assays are preferably used for the analysis of feeds and tissue samples and meats from domestic animals, particularly bovine, porcine, and ovine, most particularly, cattle, swine, and sheep.
Another application of the monoclonal antibodies is affinity purification of tilmicosin. The antibodies may be bound to a matrix, column, or other support using well-known techniques and used to recover or remove tilmicosin from any desired material. Alternatively, the monoclonal antibodies may be incorporated into sensors such as solid phase electronic devices for detection of tilmicosin in sample materials.